A novel subtilisin-related serine proteinase (ALP2) [EC 3.4.21.48] with a broad range of activity between pH 4.5 and 11.0 was released from a cell wall fraction of Aspergillus fumigatus by an alkaline pH shift. The enzyme which was not detected in the culture supernatant was partially purified by phenylbutylamine agarose chromatography. The N-terminal sequence revealed that ALP2 is the same protein identified as the major allergen of A. fumigatus in patients suffering from extrinsic bronchial asthma (Shen et al. 1999, Int. Arch. Allergy Immunol. 119, 259-264). Based on this N-terminal sequence and on a conserved region of fungal subtilisins, a specific PCR probe was generated and the ALP2 genomic and cDNA were isolated from corresponding phage libraries. ALP2 shares a 49% identity with the vacuolar proteinase B (PrB) of Saccharomyces cerevisiae. In addition there is a 78% identity with PEPC, a serine proteinase which has been described in Aspergillus niger. Targeted disruption of the ALP2-encoding gene resulted in a slightly decreased speed of vegetative growth and in a more than 80% reduction of sporulation in the alp2-negative mutants, correlated with an approximately 50% reduction of the median diameter of conidiophore vesicles. The requirement of ALP2 for regular sporulation, in addition to its role in allergic asthma, raises further interest in cellular proteinases in respect to morphogenesis and pathogenesis in A. fumigatus.